RESUMO
The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.
Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Camundongos , Humanos , Animais , Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Camundongos Transgênicos , Caveolinas/metabolismo , Mamíferos/metabolismoRESUMO
[Figure: see text].
Assuntos
Arritmias Cardíacas/metabolismo , AMP Cíclico/metabolismo , Microscopia de Fluorescência , Imagem Molecular , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Idoso , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Técnicas Biossensoriais , Sinalização do Cálcio , Modelos Animais de Doenças , Feminino , Transferência Ressonante de Energia de Fluorescência , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Preparação de Coração Isolado , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Fatores de TempoRESUMO
The assessment of local concentrations of extracellular ATP (eATP) at the site of receptor binding remains a challenge in the field of purinergic signaling. In many cases, biosensors exploiting the principle of Förster resonance energy transfer (FRET) have provided useful tools to visualize local concentrations of metabolites. A series of FRET-based biosensors based on the epsilon subunits of bacterial ATP synthases have been described for the visualisation of ATP. These sensors carry ATP-sensing units with different affinities for ATP, permitting imaging of ATP under the widely different concentration conditions found in subcellular locations such as the cytoplasm and the membrane-proximal extracellular space.
Assuntos
Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/métodos , Membrana Celular/metabolismo , Citosol/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Monitorização Fisiológica/métodos , Células HEK293 , HumanosRESUMO
AIMS: Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium-potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. METHODS AND RESULTS: A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by ß2-adrenoceptors (ß2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific ß2-AR/cAMP signals and ß2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. CONCLUSIONS: NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by ß2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted ß2-AR signalling to cAMP in the vicinity of PLM.